Insulin-like peptide 3 (INSL3), also known as relaxin-like factor, plays a key role in testicular descent in rodents. However, the roles of INSL3 in reproductive organs after puberty are still unknown for many aspects in domesticated animals. This study was undertaken to: (1) clarify quantitative changes of testicular INSL3 and its receptor, RXFP2, during and after puberty of normal male dogs; (2) compare amounts of INSL3 and its receptor in retained testes of cryptorchid dogs with those in scrotal testes of normal dogs; and (3) localize cells expressing INSL3 in canine testes.
 Testes were obtained from small-breed normal male dogs (n=56) and cryptorchid dogs (n=22). Normal scrotal testes from the normal dogs and retained testes from the cryptorchid dogs were used for the following analyses. Concentrations of testicular INSL3 and RXFP2 mRNAs were measured by real-time PCR. Concentration of testicular INSL3 peptide was assayed by an enzyme immunoassay. Immunohistochemistry for INSL3 was done in paraformaldehyde-fixed frozen testicular tissue.
 In normal dogs, INSL3 mRNA concentrations decreased from pubertal (6 to 12 months) to post-pubertal (1 to 5 years) and from post-pubertal to middle age (5 to 10 years) (P<0.05), whereas RXFP2 mRNA increased from pubertal to post-pubertal age (P<0.01). INSL3 mRNA concentrations were higher for the retained testes in the cryptorchid dogs than for the normal testes in normal dogs (P<0.01). RXFP2 mRNA concentrations were almost negligible in the retained testes, and much lower than those in the normal testes (P<0.01). INSL3 peptide concentrations neither differed among age groups nor between normal and retained testes. Immunohistochemical analysis revealed that INSL3 is solely expressed in Leydig cells in both normal and retained testes.
 Taken together, these results suggest that RXFP2 gene expression is strikingly suppressed in retained testes of cryptorchid dogs, in spite of substantial expression of INSL3.